Oxidative stress is a major factor in Parkinson's Disease (PD). Dopamine (DA) itself is easily oxidized to quinone derivatives and reactive oxygen species (ROS) that impair energy metabolism and form adducts with proteins such as upsilon-synuclein. Because pharmacological depletion of DA in animal models is confounded by non-specific peripheral and central nervous system effects, the role of DA oxidation in nigral cell death has been previously impossible to address. Thus a key unanswered hypothesis in this field is that DA oxidation is a major contributor to the death of dopaminergic neurons in PD. The proposed studies address several aspects of this hypothesis including the interaction of known environmental factors in triggering DA oxidation. Specifically, the hypothesis that the DA-releasing potential of the parkinsonism-inducing drug, MPP+, is due to its ability to exchange with DA and/or to reduce intracellular pH gradients will be addressed using newly derived mice expressing enhanced green fluorescent protein from a dopaminergic locus (TH+/eGFP). Primary cultures derived from these animals as well purified synaptosomal and vesicular preparations from dopaminergic terminal fields will be used in combination with fluorescent and radioactive probes to determine the temporal aspects of DA release, intracellular membrane changes, ROS formation, ATP loss, etc in response to toxin treatment. In addition, the hypothesis that DA oxidation contributes to the death of dopaminergic cells will be directly tested in vivo using animals genetically engineered to have different levels of DA production. Behavioral, oxidative and immunocytochemical criteria will be used to establish the role of DA in both the acute and chronic MPTP model of PD. To test whether DA depletion prevents ROS, new methodologies to detect in situ ROS will be used with a battery of antibodies directed against nitrotyrosine, nitrated alpha-synuclein, etc. to temporally evaluate ROS formation following acute or chronic MPTP administration in DA deficient and wild type animals. Taken together, the proposed studies will determine whether DA oxidation plays a central role in the death of DA synthesizing cells and provide insights impossible to obtain from standard animal models. Knowledge of the source and cascade of events surrounding DA-induced free radical formation will help answer risk-benefit controversies surrounding the use of dopamine replacement therapies as well as facilitate the development of new drugs and/or treatment strategies in the pathogenesis of PD.